Synthesis of acridine derivative multidrug-resistant inhibitors

ABSTRACT

Synthesis of the MDRI of formula (I) from intermediates of acridone acid of formula (II) and aminophenethyl isoquinoline of formula (III), via steps including coupling and conversion to yield the MDRI of formula (I).

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application No.PCT/EP98/02991 filed May 22, 1998, which claims priority from GB9710612.4 filed May 23, 1997.

BACKGROUND OF THE INVENTION

This invention relates to an improved process for preparing acridinederivatives. In particular it relates to the synthesis of compoundswhich are capable of sensitizing multidrug-resistant cancer cells tochemotherapeutic agents.

The multidrug-resistant inhibitor (MDRI), chemically known asN-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide and its physiologically acceptable salts is described inWorld Patent Application WO 92/12132, filed in the name of LaboratoriesGlaxo S.A. and published Jul. 23, 1992 and also described in WorldPatent Application WO 96/11007, filed in the name of Glaxo Wellcome Inc.and published Apr. 18, 1996. The compounds are disclosed as being usefulin sensitizing multidrug-resistant cancer cells to chemotherapeuticagents.

In WO 92/12132, an acridine derivative was disclosed as synthesized byreacting compounds in the presence of coupling reagents commonly used inpeptide synthesizing. The coupling reagents disclosed includeddicyclohexylcarbodiimide (optionally in the presence of1-hydroxybenzotriazole), diphenylphosphoryl azide or N,N-carbonyidiimidazole. Suitable inert solvents for the reaction includedan ether, halogenated hydrocarbons, amides or ketones.

The synthesis ofN-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo4-acridinecarboxamide and its physiologically acceptable salts and solvates isalso disclosed by Ne'rina Dodic et al., Journal of Medicinal Chemistry,1995, Vol. 38 No. 13, pages 2418-2426. The synthesis route in Dodicutilized the same coupling reagents as set forth in WO 92/12132. In theDodic article, example 84 corresponds toN-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide.

SUMMARY OF THE INVENTION

The present invention provides an improved process of synthesizing themultidrug-resistant inhibitor, hydrochloride salt ofN-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide. This process eliminates the use of prior art couplingreagents which produced a water insoluble diisopropyl urea by-productduring the coupling of the intermediates. This urea by-product was noteasily removed. The prior art also suggested using a chlorinatedsolvent, i.e., dichloromethane, during the intermediate synthesisstages.

The present invention further provides an improved process wherein theby-product, tetramethyl urea, formed from the coupled intermediates iswater soluble and easily removed. Furthermore, the present inventiveprocess eliminates the use of chlorinated solvents and allows for directcrystallization of the intermediates from the reaction mixture.

The present invention further provides an improved process havingincreased throughput and products having higher purity.

The present invention includes synthetic steps and intermediatesinvolved in a scheme of synthesizing the hydrochloride salt,N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide of the following formula (I)

DETAILED DESCRIPTION OF THE INVENTION

In the description and examples that follow throughout thespecification, the following abbreviations may be used: THF(tetrahydrofuran); DMF (N,N-dimethyl formamide); TBTU(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate); DMSO (dimethylsulfoxide); g (grams); mL(milliliters); mp (melting point); ¹H-NMR (proton nuclear magneticresonance); ppm (parts per million); MHz (Megahertz); and eq. (molarequivalents). Unless otherwise noted, all temperatures are expressed in° C. (degrees centigrade).

¹H-NMR spectra were measured in DMSO using a Bruker ARX-300 MHzinstrument. Chemical shifts are expressed in ppm in reference to aninternal standard such as DMSO. Apparent multiplicities are designateds, singlet; d, doublet; t, triplet; m, multiplet; br s, broad singlet.Melting points were determined on a Perkin Elmer DSC 7. HPLC data wascollected on a Hitachi L-6200 A pump, L-4000 UV detector and a D-2500integrator.

The materials used in the synthesis process are available from AldrichChemical Company, which is located in Milwaukee, Wisconsin. The peptidecoupling reagent, TBTU, is available from Peboc, Llangefui, Anglesey,Gwynedd, which is located in Wales, UK. The filtering aid, Harborlite,is available from Harborlite, which is located in Hull, UK.

The synthesis process is carried out in the presence of couplingreagents used in peptide synthesis, such as tetramethyluronium saltbased peptide coupling agents and tetramethyluronium salt based acidactivating agents. Exemplary agents include, TBTU,O-(Benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,O-(7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate andO-(1,2-Dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate. Other acid activating reagents, such as1,1′-carbonyidiimidazole, can be utilized in the synthesis process. Thesynthesis process can be carried out in a alkylamine base, such astriethylamine and diisopropylamine in addition to aromatic amine basessuch as pyridine. Suitable solvents for the synthesis process includepolar aprotic solvents, such as DMF or 1-methyl-2-pyrrolidinone as wellas acetonitrile.

The starting compounds of Scheme 1, to prepare the compound of formula(II), are prepared in accordance with stages 1 and 2 below:

In stage 1, the methoxydiacid of formula (IIc) is obtained by forming asuspension of an 2-amino-3-methoxybenzoic acid (IIa), 2-bromobenzoicacid (IIb) potassium carbonate and copper powder which is stirred inethanol and heated to reflux for at least 0.5 hours, preferably 1 hour.The suspension is cooled to 20-25° C. and water added. A filtering aidis added and the mixture filtered. The filter bed is washed with waterand the combined filtrates adjusted to pH 2-3 by the addition ofconcentrated hydrochloric acid over a period of about 30 minutes. Theresulting suspension is then aged in the reactor at 10-12° C. for atleast 1 hour and the solid product collected by filtration, washed withwater and dried in a vacuum at 50° C.

In stage 2, the methoxydiacid of formula (IIc), formed in stage 1 ismixed with acetonitrile and heated at reflux and phosphorous oxychlorideis added dropwise over 2 hours. The resulting mixture is heated atreflux for at least 1 hour preferably 2 hours. This mixture is thencooled to 10-15° C. Water is added and the resultant thick slurry isheated at reflux for 2.5 hours. The slurry is then cooled to 10° C. andfiltered. The product, acridone acid of formula (II), is washed withwater followed by acetonitrile and dried in vacuum at 50° C. for 48hours.

The starting compounds of Scheme 1, to prepare the compound of formula(III), are prepared in accordance with stages 3 and 4 below

In stage 3, the nitrophenethyl isoquinoline of formula (IIIc) isobtained by mixing 4-nitrophenethyl bromide, of formula (IIIa),6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, of formula(IIIb), anhydrous potassium carbonate and potassium iodide in DMF. Themixture is heated at 70° C. with stirring under a nitrogen atmospherefor 18 hours. The mixture is cooled to 50° C. and methanol added. Themixture is further cooled to 30° C. and water added. The mixture is thenstirred at 10° C. for 1 hour, filtered, and the product washed withwater and dried at 45° C. under vacuum

In stage 4, the nitrophenethyl isoquinoline, or formula (IIIc), formedin stage 3 is stirred in a solution of ethanol and THF at 15-20° andpurged with nitrogen and a Pd/C catalyst added. After re-purging withnitrogen, the stirring is stopped and the mixture is purged withhydrogen. Stirring is resumed and the mixture maintained at 15-25° C.until hydrogen uptake is complete. The reaction mixture is filtered, thefilters rinsed with THF and the combined filtrates concentrated to anappropriate volume under vacuum at 55-65° C. Hexane is added over a 2040minute period and the resulting slurry cooled to 0° C. After stirring at0° C. for 1.5 hours, the suspension is filtered, the solid washed withhexane and dried in a vacuum oven at 40-45° C. The resulting solid isaminophenethyl isoquinoline of formula (III).

The intermediate compounds of scheme 1, to prepare the multidruginhibitor free base compound of formula (IV) is prepared in accordancewith stage 5.

In stage 5, Scheme 2, a mixture of the intermediate compounds, acridoneacid of formula (II) and aminophenethyl isoquinoline of formula (III)are stirred in DMF under a nitrogen atmosphere until completedissolution is accomplished in about 10 minutes. A peptide couplingreagent, TBTU, is added followed by triethylamine, a base. The solutionis stirred at 20-25° C. for 1-2 hours until the reaction is complete. Amixture of isopropanol-water is added and the mixture stirred at 20-25°C. for 30-60 minutes until crystallization occurs. The resulting slurry,MDRI free base of formula (IV), is filtered and washed with methanol,followed by water and dried in a vacuum oven at up to 50° C.

The crude MDRI free base is then recrystallized by dissolving in DMF at35-40° C. followed by the addition of ethanol over a period of about 4hours at 35-40° C. The resulting slurry is then cooled at 10° C. for 1hour and filtered. The product is washed with methanol and dried in avacuum oven at up to 50° C.

The compound of formula (I) in scheme 2, wherein the MDRI free base offormula (Iv) is converted into a hydrochloride salt of formula (I) inaccordance with stage 6.

In stage 6, a stirred suspension of the MDRI free base of formula (Iv)in glacial acetic acid is heated to 65-70° C. and the resulting solutionhot filtered. The solution is then reheated to 70° C. and hot,pre-filtered ethanol is added. Pre-filtered concentrated hydrochloricacid is then added over a period of about 30 minutes. The resultingsolution is stirred at 70° C. until crystals form, about 20 minutes, andthen cooled to 20-25° C. over 1 hour and filtered. The resulting filtercake is washed with ethanol and dried for at least 70 hours at 65° C. ina vacuum oven.

EXAMPLES Example 1 Methoxydiacid (Formula IIc)

A suspension of 164.6 g (1 molar eq) of 2-amino-3-methoxybenzoic acid(formula IIa), 217.7 g (1.1 molar eq) of 2-bromobenzoic-acid (formulaIIb), 272.3 g (2.0 molar eq) of potassium carbonate and 12.5 g (0.2molar eq) of copper powder is stirred in 1500 mL of ethanol and heatedto reflux for 0.5-3 hrs, preferably 1 hour. The suspension is cooled to20-25° C. and 1450 mL of water is added. A filtering aid, 15 g, is addedand the mixture filtered. The filter bed is washed with 875mL of waterand the combined filtrates adjusted to a pH of 2-3 by the addition of250 mL of concentrated hydrochloric acid over a period of about 30 min.The resulting suspension is then aged in the reactor at 10-12° C. for atleast 1 hour and the solid product collected by filtration, washed with1450 mL of water and dried in a vacuum at 50° C. The expected yield ofthe titled compound is 95% theoretical.

¹H-NMR (300 MHz): δ 3.7 (s, 3H, OMe); 6.1-7.9 (m, 7H, Ar); 9.7 (br s,1H, COOH); 11.1 (br s, 1H, COOH) ppm. Mp is 254-256° C.

Example 2 Acridone Acid (Formula II)

A mixture of 280.0 g (1 molar eq) of methoxydiacid (formula IIc) in 2300mL of acetonitrile is heated at reflux and 200 mL (2.2 molar eq) ofphosphorous oxychloride added dropwise over 2 hours. The mixture isheated at reflux for 1-5 hours, preferably 2 hours. The mixture is thencooled to 10-15° C. To the mixture 1700 mL of water is added and theresultant thick slurry is heated at reflux for 2.5 hours. The slurry isthen cooled to 10° C. and filtered. The resulting product is washedtwice with 850 mL of water, washed twice with 850 mL of acetonitrile anddried in vacuum at 50° C. for 48 hours. The expected yield of the titledcompound is 95% theoretical.

¹H-NMR (300 MHz): δ 4.0 (s, 3H, OMe); 6.9-8.5(m, 7H, 6Ar and NH); 10.1(br s, 1H, COOH) ppm. Mp is 354-362° C.

Example 3 Nitrophenethyl Isoquinoline (Formula IIIc)

A mixture of 302 g (1 molar eq) of 4-nitrophenethyl bromide (formulaIIIa), 302 g (1 molar eq) of6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (formulaIIIb), 181 g (1.1 molar eq) of anhydrous potassium carbonate and 45 g(0.2 molar eq) of potassium iodide in 1500 mL of DMF is heated at 70° C.with stirring under a nitrogen atmosphere for 12-24 hours, preferably 18hours. The mixture is cooled to 50° C. and 450 mL of methanol is added.The mixture is then cooled to 30° C. before adding 3000 mL of water. Themixture is stirred at 10° C. for 1 hour, filtered and the product washedtwice with 1500 mL of water and dried at 45° C. under vacuum. Theexpected yield of the titled compound is 90% theoretical.

¹H-NMR (300 MHz): δ 2.53-3.0 (m, 8H, CH₂); 3.5 (s, 2H, N—CH₂—Ph); 3.7(s, 6H, Ome); 6.4 (d, 2H, Ar isoquinoline); 7.2 and 7.9 (dd, 4H, ArPHNO₂) ppm. Mp is 116-118° C.

Example 4 Aminophenethyl Isoquinoline (Formula III)

A stirred solution of 230.0 g (I molar eq) of nitrophenethylisoquinoline (formula IIIc) in 1700 mL of ethanol and 1700 mL of THF at15-20° C. is purged with nitrogen and 46 g of Pd/C catalyst is added.After re-purging with nitrogen, the stirring is stopped and the mixtureis purged with hydrogen. Stirring is resumed and the mixture ismaintained at 15-25° C. until hydrogen uptake is complete (1-20 hours).The reaction mixture is filtered, the filter is rinsed with 900 mL ofTHF and the combined filtrates are then concentrated to 7 volumes undervacuum at 55-65° C. To the concentrated filtrate is added 2000 mL ofhexane over 20-40 minutes, and the resulting slurry cooled to 0° C.After stirring at 0° C. for 1.5 hours the suspension is filtered, thesolid washed with 450 mL of hexane and dried in a vacuum oven at 40-45°C. The expected yield of the titled compound is 89% theoretical.

¹H-NMR (300 MHz) δ 2.5-3.0 (m, 8H, CH₂); 3.5 (s, 2H, N—CH₂—Ph); 3.7 (s,6H, OMe); 6.4 (d, 2H, Ar isoquinoline); 7.2 and 7.9 (dd, 4H, Ar PHNO₂)ppm. Mp is 123-126° C.

Example 5 MDRI Free Base-(Formula IV)

A mixture of 200.0 g (1 molar eq) of acridone acid (formula II) and232.1 g (1 molar eq) of aminophenethyl isoquinoline (formula III)stirred in 2000 mL of DMF at 20-25° C. until complete dissolution isaccomplished in about 10 minutes. To this mixture 250.5 g (1.05 molareq) of TBTU, a peptide coupling reagent, is added, followed by 218 mL(2.1 molar eq) of triethylamine, a base. The resulting solution isstirred at 20-25° C. for 1-2 hours until the reaction is complete. A 1:1mixture of 12000 mL of isopropanol and 1000 mL of water is added and themixture stirred at 20-25° C. until crystallization occurs (30-60minutes). The resulting slurry is filtered and washed with 1600 mL ofmethanol, followed by washing with 1600 mL of water. The slurry is driedin a vacuum oven at up to 50° C.

The crude MDRI free base (formula IV) is then recrystallized bydissolving in 1800 mL of DMF at 35-40° C., followed by the addition of3600 mL of ethanol over a period of about 4 hours at 35-40° C. Theresulting slurry is then cooled to 10° C. for 1 hour and filtered. Theproduct is washed with 1000 mL of methanol and dried in a vacuum oven atup to 50° C. The expected yield of the titled compound is 70-75%theoretical.

¹H-NMR (300 MHz): δ 2.40-2.95 (m, 8H, CH₂); 3.58 (s, 2H, N—CH₂—Ph); 3.72(s, 6H, 2OMe); 4.05 (s, 3H, OMe acridone); 6.78 (d, 2H, Arisoquinoline); 7.20-7.88 (m, 8H, Ar); 8.48 (t, 2K, H₂ and H₇ acridone);10.60 (br s, 1H, CONK); 12.32 (br s, 1H, NH acridone) ppm. Mp is215-220° C.

Example 6 MDRI Drug Substance (Formula I)

A stirred suspension of 20.0 g (1 molar eq) of MDRI free base (formulaIV) in 80 mL of glacial acetic acid is heated to 65-70° C. and theresulting solution hot filtered. The solution is then reheated to 70° C.and 240 mL of pre-filtered ethanol (70° C.) is added. To this solution,4.4 mL (1.5 molar eq) of pre-filtered concentrated hydrochloric acid isadded over a period of about 30 minutes. The resulting solution isstirred at 70° C. until crystals form, about 20 minutes, and cooled to20-25° C. over a 1 hour period and filtered. The resulting filter cakeis twice washed with 120 mL of ethanol and dried for at least 70 hoursat 65° C. in a vacuum oven. The expected yield of the titled compound is90% theoretical.

¹H-NMR (500 MHz, DMSO-d₆) δ 12.31 (s, 1H), 10.98 (brs, 1H), 10.75 (s,1H), 8.53 (dd, 1H), 8.52 (ddd, 1H), 7.82 (ddd, 1H), 7.76 (m, 2H), 7.44(dd, 1H), 7.40 (dd, 1H), 7.38 (m, 2H), 7.28 (dd, 1H), 6.84 (s, 1H), 6.81(s, 1H), 4.52 (d, 1H), 4.29 (dd, 1H), 4.06 (s, 3H) 3.77 (m, 1H), 3.76(s, 3H), 3.75 (s, 3H), 3.45 (m, 2H), 3.33 (m, 1H), 3.19 (m, 1H), 3.19(m, 2H), 2.96 (dt, 1H) ppm. Mp is 240° C.

Anal. Calc'd. for C₃₄H₃₃N₃O₅.HCl.0.5H₂O: C, 67.04; H, 5.57; N, 6.90; Cl,5.82;

Found: C, 67.00; H, 5.78; N, 6.89; Cl, 5.87.

What is claimed is:
 1. A method of synthesizing a compound of formula(IV)

which comprises the steps of: (i) dissolving a compound of formula (II):

and a compound of formula (III):

in a polar aprotic solvent; (ii) reacting said compounds of formula (II)and (III) dissolved in said solvent in the presence of atetramethyluronium based peptide coupling reagent and an alkylamine toform a product mixture; and (iii) crystallizing directly from theproduct mixture a product comprising a free base of formula IV; (iv)optionally converting the compound of formula (IV) to the HCl salt offormula (I)


2. The method of claim 1, wherein the coupling reagent is2-(1H-benzotriazole-1-yl)-1,1,3, 3-tetramethyluronium tetrafluoroborate.3. The method of claim 1, wherein the alkylamine base is triethylamine.4. The method of claim 1, wherein the reaction takes place at about20-25° C.
 5. The method of claim 1, wherein the reaction takes place ina period of at least one hour.
 6. The method of claim 1, wherein saidcoupling step is conducted at about 20-25° C.
 7. The method of claim 1,wherein the polar aprotic solvent is dimethyl formamide.
 8. The methodof claim 1, wherein the compound of formula IV is converted to the HClsalt of formula I.